(E)-2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate ((E)-FMCDP), obtained by phosphorylation of the clinically promising anti-tumor agent MDL 10 1,73 1, is a potent time-dependent inactivator of RNR. EPR spectroscopy reveals that inactivation is accompanied by loss of Tyro and formation of a new radical species. The 9.4 GHz EPR spectrum of this new radical reveals two hyperfine splittings of approximately 1.5 mT, producing a triplet like signal. Incubation with [6'-2H](E)-FMCDP alters this EPR spectrum, providing evidence for a nucleotide based radical species generated by RDPR. The observed signal is a 1: 1 composite of a doublet and a triplet signal, the latter being identical to that obtained with unlabeled inhibitor. Studies with (E)-FMCDP in 2H20 also produce a 1: 1 mixture of these two radical signals. The results of these isotope labeling experiments [unreadable]suggest wash-out/in of -0.5 equivalents of deuterium at the 6-position, respectively. Incubation of RNR with [6'-2H]-(E)-FMCDP produced only the doublet as this hypothesis leads us to expect. 139.5 GHz EPR spectra established the principal g-values of the new radical species. Simulation of the 9.4 and 139.5 GHz EPR spectra yield a self-consistent set of principal hyperfine values. The radical intermediate is proposed to arise from a substrate based allyl radical. The proposed structure and a postulated mechanism for its formation provides further support for the hypothesis that catalysis is initiated by Y-hydrogen atom abstraction from the nucleotide substrate.